Method of treating ulcerative colitis

ABSTRACT

A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon which contains in combination (a) an oil blend which contains eicosapentaenoic acid (20:5n3) and/or docosahexaenoic acid (22:6n3), and (b) a source of indigestible carbohydrate which is metabolized to short chain fatty acids by microorganisms present in the human colon. Preferably the nutritional product also contains one or more nutrients which act as antioxidants.

The present invention relates to a method of treating a mammaliancreature, preferably a human, having ulcerative colitis or inflammationof the colon.

The term "Inflammatory Bowel Disease" is a designation commonly used fortwo related, but distinct, chronic inflammatory conditions affecting thegastrointestinal tract, namely Crohn's disease and ulcerative colitis.Crohn's disease may involve any segment of the gastrointestinal tract,although characteristically the region of greatest involvement is thedistal one quarter of the small intestine and the proximal colon. Inulcerative colitis the inflammation is, by definition, limited to themucosa of the large bowel. However, the present invention is concernedonly with nutritional support for a person having ulcerative colitis.The primary cause of ulcerative colitis is not currently known.

At the present time, there is no medical cure for ulcerative colitis andthis chronic condition may lead to total proctocolectomy. Currentmedical treatment is directed toward decreasing the number, frequencyand severity of acute exacerbations of inflammatory bowel disease andpreventing secondary complications, but at best, the results aredisappointing. Long term use of corticosteroids to downregulate theinflammatory response is a common approach to the control of intestinalinflammation. Steroids are considered to exert their antiinflammatoryeffects through inhibition of the release of free arachidonic acid frommembrane phospholipids. Historically the long term use ofimmunosuppressive agents (steroids) is associated with chronic sideeffects such as those presented in Table 1.

Sulfasalazine is widely used to treat victims of ulcerative colitis.Sulfasalazine's pharmacologic effects include alterations in thebacterial flora of the gut, increased colonic absorption of fluids andelectrolytes, decreases in the number of B cells, interference withlymphocyte activation and natural killer activity, and inhibition ofantibody secretion. The overall usefulness of sulfasalazine has beensomewhat undermined by a high degree of intolerance and a frequentoccurrence of adverse reactions in the patient population such as thosepresented in Table 1.

                  TABLE 1                                                         ______________________________________                                        CURRENT DRUG THERAPIES                                                        FOR ULCERATIVE COLITIS                                                        "non-specific therapies"                                                                   AD-                                                              DRUG         MINISTRATION  SIDE EFFECTS                                       ______________________________________                                        Anti-inflammatory agents                                                      Salicylates  oral, rectal  secretory diarrhea,                                5-ASA (ROWASA)                                                                             (enemas)      nausea, headache,                                  Sulfasalazine              anemia, leukopenia                                 Corticosteroids                                                                            oral, topical,                                                                              acne, weight gain,                                              intravenous   peptic ulcer,                                                                 diabetes, glaucoma,                                                           cataracts,                                                                    osteoporosis,                                                                 psychosia                                          Imunosuppressive agents                                                       Azathioprine (AZA)                                                                         oral, intravenous                                                                           bone marrow                                        6-mercaptopurine           suppression,                                       Metronidazole              infections,                                        Cyclosporine               pancreatitis                                       Prednisone                                                                    ______________________________________                                    

Antibiotics are used intermittently, particularly in the presence ofsevere exacerbations as are other drugs including antispasmodics andanticholinergics. It has been reported by Rosenberg et al., "NutritionalAspects of Inflammatory Bowel Disease", ANNUAL REVIEW OF NUTRITION.,Vol. 5, pages 463-484, at 467 (1985) that many drug therapies used ininflammatory bowel diseases may have negative effects on nutritionalstatus. For example, high daily doses of corticosteroids can exert anadditional catabolic effect on patients who may already be under stress,and may inhibit calcium absorption by the intestine. Another example ofa potentially negative drug-nutrient interaction is the interferencewith folate absorption by sulfasalazine via a mechanism of competitiveinhibition.

Therapy for severe attacks of ulcerative colitis frequently includesspecial nutritional support, especially when surgical intervention isplanned. Total parenteral nutrition was initially used to improvenutritional status, but later was used to enhance "bowel rest" andinduce clinical remission to avoid total proctocolectomy. However;Gonzalez-Huix et al., "Enteral versus Parenteral Nutrition as AdjunctTherapy in Acute Ulcerative Colitis", THE AMERICAN JOURNAL OFGASTROENTEROLOGY, Vol. 8, No. 2, pages 227-232 (1993) reports theresults of a study which suggests that total enteral nutrition is safeand nutritionally effective in severe attacks of ulcerative colitis.This publication suggests total enteral nutrition should be regarded asthe most suitable type of nutritional support in these patients. Theenteral nutritional product used in this published study was Edanec HNfrom UNIASA, Granada, Spain which was described in the publication asset forth below in Table 2.

                  TABLE 2                                                         ______________________________________                                        EDANEC HN                                                                     NUTRIENT           AMOUNT PER 1000 ml                                         ______________________________________                                        Nitrogen (g)       8.73                                                       Lipids (g)         36.20                                                      Carbohydrates (g)  110.20                                                     Energy (Kcal)      984.85                                                     E/N ratio (nonprotein kcal/g N)                                                                  87.81                                                      Energy source                                                                 Nitrogen           Intact milk protein                                        Fat                Long Chain Triglycerides                                   Carbohydrate       Maltodextrins                                              Na (mol)           36.00                                                      K (mmol)           32.00                                                      Calcium (mmol)     3.00                                                       Magnesium (mmol    3.00                                                       Phosphate (mmol)   12.00                                                      Vitamins           Upper limit of RDA                                         Trace-elements     Upper limit of RDA                                         ______________________________________                                    

Gonzalez-Huix et al., compared the effects of total enteral nutritionand total parenteral nutrition in patients with acute ulcerativecolitis. The final conclusions of their trials were that totalparenteral nutrition does not have a primary therapeutic effect on theinflammatory process, and that "bowel rest" is not essential for themanagement of acute ulcerative colitis. The main reluctance to useenteral feeding in severe ulcerative colitis has been the possibility ofworsening diarrhea. Gonzalez-Huix et al. reported that only one patientout of 23 fed enterally developed diet-related diarrhea. Although aregular diet may be well-tolerated in ulcerative colitis, patients tendto reduce food intake unless they are persistently encouraged to eat. Inthese circumstances, tube feeding has been used to guarantee adequateenergy and nutrient supply.

The UNIASA product, Edanec HN, differs considerably from the nutritionalproduct of the present invention. For example, the new product of thepresent invention has a caloric density of 1.29 kcal/ml while Edanec HNhas a caloric density of 0.98 kcal/ml. Our product also is lower in fat,containing approximately 21.9 g Fat/1000 kcal while Edanec HN containsapproximately 36.7 g Fat/1000 kcal. The nutritional product of thepresent invention also contains fish oil as a source of eicosapentaenoicacid (20:5n3) and docosahexaenoic acid (22:6n3) as well as dietaryfibers such as gum arabic and indigestible oligosaccharides such asfructooligosaccharides (FOS) and xylooligosaccharides (XOS). Theseingredients are crucial for a product developed for a patient withulcerative colitis.

Ulcerative colitis afflicts persons as young as 5 years old. Onset ofsymptoms of inflammatory bowel disease occurs before age 20 in about 40%of patients. The biggest problem in the management of ulcerative colitisin young persons is almost invariably poor dietary compliance. It hasbeen observed by Sutton, "Nutritional Needs of Children withInflammatory Bowel Disease", NUTRITION, Vol. 18, No. 10, pages 21-25(1992) that deficiencies of micronutrients are individually determinedand relate to disease activity and site as well as dietary intake.Sutton recommends a multivitamin/mineral tablet which meets 100-150% ofthe Recommended Daily Allowance. This publication further reports that:(a) deficiencies of water-soluble nutrients such as folate, B₁₂, biotin,vitamin C, niacin, riboflavin, and B₆ have been reported in patients whoeliminated foods such as milk, fruits and vegetables due to intolerance;(b) deficiencies of fat-soluble nutrients such as vitamins A, E and Khave been reported in patients having fat malabsorption due to severeileac disease or resection; and (c) deficiencies of minerals and traceminerals such as calcium, iron, zinc, copper and chromium, result frominadequate intake and/or reduced absorption.

Similar nutritional deficiencies in inflammatory bowel disease patientshave been reported by Rosenberg et al., "Nutritional Aspects ofInflammatory Bowel Disease", ANNUAL REVIEW OF NUTRITION, Vol. 5, pages463-484 (1985). Rosenberg, et al. describe the problems of proteincalorie malnutrition and deficiency of micronutrients ongastrointestinal function and structure in these terms: The patient withinflammatory bowel disease who becomes significantly malnourished mayenter a vicious cycle where secondary effects of malnutrition orgastrointestinal function and structure may lead to a further increasein gastrointestinal symptoms and malabsorption, which further worsensnutrient balance. In addition, it may be assumed that malnutrition willsignificantly depress the patient's ability to heal the inflammation andstructural changes in the bowel. The overall therapeutic strategy mustbe to ensure adequate intake of nutrients while modifying dietary intaketo decrease gastrointestinal symptoms.

The impact of ulcerative colitis on nutritional status can be highlysignificant, particularly in the pediatric age group, in whom proteinand calorie requirements for growth are not likely to be met by ordinarydietary means. There is increasing evidence that a good therapeuticresponse can be achieved in ulcerative colitis by dietary treatmentalone. Many dietary regimens have fallen short of expectations and havenot been uniformly effective in promoting weight gain and wound healingor in maintaining optimal nutritional status in patients with ulcerativecolitis.

The features of the present invention which are believed to be novel areset forth with particularity in the appended claims. The presentinvention may be understood by referring to the following detaileddescription, taken in accordance with the accompanying drawing FIGS. 1-5which are all charts presenting the results of experiments relating tothe present invention.

The major advantages of using a specially formulated enteral diet toinduce remission of active disease include the virtual absence of sideeffects, possible decreased dosage of prescribed drugs and improvednutritional status of adults and children. In order to understand andevaluate the effects of polymeric diet(s), various nutrients such as n-3fatty acids, nutrients which function as antioxidants, and short chainfatty acids (SCFAs) must be assessed as to their ability to decreasedisease activity in ulcerative colitis and allow for mucosal repair.

Increasing interest has been generated in the use of enemas/irrigationsolutions containing buffered, physiologic levels of SCFAs for thetreatment of diversion colitis and ulcerative colitis. Diversion colitisis an inflammatory process arising in segments of the colorectum atvarious intervals after surgical diversion of the fecal stream. Theendoscopic appearance is similar to those of active Crohn's Disease andulcerative colitis. Glotzer et al., "Proctitis and Colitis FollowingDiversion of the Fecal Stream", GASTROENTEROLOGY Vol. 80, pages 438-441(1981). The cause of this condition is not known, but one mechanism hasbeen postulated; a nutritional deficiency of the colonic epithelium,specifically due to the absence of SCFAs normally present in coloniccontents, Komorowski, "Histologic Spectrum of Diversion Colitis"AMERICAN JOURNAL OF SURGICAL PATHOLOGY, Vol. 14, page 548 (1990),Roediger, "The Starved Colon--Diminished Mucosal Nutrition, DiminishedAbsorption, and Colitis", DISEASES OF THE COLON AND RECTUM, Vol. 33,pages 858-862 (1990). Harig et al., "Treatment of Diversion Colitis withShort-Chain-Fatty Acid Irrigation", NEW ENGLAND JOURNAL OF MEDICINE,Vol. 320 No. 1, pages 23-28 (1989) tested this hypothesis by assessingwhether irrigation with SCFAs could ameliorate inflammation in fourpatients with diversion colitis. These patients were administered SCFAstwice daily for 2-3 weeks with 60 mL of an enema solution comprising aphysiologic mixture of SCFAs as sodium salts. After 2-3 weeks oftherapy, macroscopic and histological resolution of inflammation wasevident. An impaired utilization of SCFAs has also been implicated inulcerative colitis which suggests that diminished intracellular energyproduction may be important in the inflammatory process, Roediger, "TheColonic Epithelium in Ulcerative Colitis: an Energy DeficiencyDisease?", THE LANCET, Oct. 4, 1980, pages 712-715 (1980). Vernia etal., "Fecal Lactate and Ulcerative Colitis", GASTROENTEROLOGY, Vol. 95,pages 1564-1568; and Vernia et al., "Organic Anions and the Diarrhea ofInflammatory Bowel Disease", DIGESTIVE DISEASES AND SCIENCES, Vol. 33,pages 1353-1358 (1988) have shown that fecal water from patients withulcerative colitis contains reduced concentrations of SCFAs as well asmarkedly increased lactate and low pH. In a study by Breuer et al.,"Rectal Irrigation with Short-Chain Fatty Acids for Distal UlcerativeColitis" (preliminary report), DIGESTIVE DISEASES AND SCIENCES, Vol. 36,pages 185-187 (1991), relates an investigation of large bowel irrigationwith SCFAs in patients with ulcerative colitis. It was found that 9 outof 10 patients completing the study were judged to be at least muchimproved and showed a significant change in mean disease activity indexscore and mucosal histology score. Recently Senagore et al.,"Short-Chain Fatty Acid Enemas: a Cost Effective Alternative in theTreatment of Nonspecific Proctosigmoiditis", DISEASES OF THE COLON ANDRECTUM, Vol. 35, page 923 (1992), confirmed the results of Breuer et al.demonstrating an 80 percent response rate in patients with idiopathicproctosigmoiditis. This study indicates that administering a solution ofSCFAs similar to Harig et al. for six weeks was equally efficacious tocorticosteroid or 5-ASA enemas for the treatment of proctosigmoiditis ata significant cost savings. Scheppach et al., "Effect of Butyrate Enemason the Colonic Mucosa in Distal Ulcerative Colitis", GASTROENTEROLOGY,Vol. 103, pages 51-56 (1992) investigated the use of butyrate enemasalone rather than the SCFA mixture to treat ten patients with distalulcerative colitis in a placebo-controlled, single-blind, randomizedtrial. The authors concluded that markedly improved disease activityindex and histological parameters suggesting that the effect of a SCFAmixture on the inflamed mucosa in ulcerative colitis is largelyattributable to its butyrate moiety.

It is unlikely that short chain fatty acids added directly to an enteralproduct would reach the large bowel. Also, the stability of thesecompounds in a nutritional product is questionable. However, thenutritional product of the present invention takes advantage of thepositive effect of SCFAs by providing dietary fiber or indigestibleoligosaccharides.

For the purpose of the patent the following terms are defined asfollows:

Dietary Fiber--A material that contains a large carbohydrate moiety(Degree of polymerization greater than 20 and/or a molecular weightgreater than 3,600) that is resistant to endogenous digestion in thehuman upper digestive tract.

Indigestible Oligosaccharide--A small carbohydrate moiety (Degree ofpolymerization less than 20 and/or a molecular weight less than 3,600)that is resistant to endogenous digestion in the human upper digestivetract.

Indigestable Carbohydrate--A term used to encompass both dietary fiberand indigestible oligosaccharides.

Certain of the organisms that inhabit the large bowel can utilizedietary fiber (eg, pectin and gum arabic) and indigestibleoligosaccharides (eg, fructooligosaccharides and xylooligosaccharides)as an energy source. Smith et al., "Introduction to Metabolic Activitiesof Intestinal Bacteria", AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol.32, pages 149-157 (1979); Miller et al., "Fermentation by SaccharolyticIntestinal Bacteria", AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol. 32,pages 164-172 (1979); Cummings, "Fermentation in the Human LargeIntestine: Evidence and Implications for Health", THE LANCET, May 28,1983, pages 1206-1209; Titgemeyer et al., "Fermentability of VariousFiber Sources by Human Fecal Bacteria In Vitro", AMERICAN JOURNAL OFCLINICAL NUTRITION, Vol. 53, pages 1418-1424 (1991). The microorganismsderive energy from the carbohydrate sources through a process referredto as anaerobic fermentation. During fermentation, the microorganismsproduce SCFAs (eg, acetate, propionate, butyrate) as the majorendproducts. Salyers et al., "Fermentation of Mucin and PlantPolysaccharides by Strains of Bacteroides from the Human Colon", APPLIEDAND ENVIRONMENTAL MICROBIOLOGY, Vol. 33, No. 2, pages 319-322 (1977);Mitsuoka et al., "Effect of Fructo-oligosaccharides on IntestinalMicroflora", DIE NAHRUNG, Vol. 31, pages 427-436 (1987); Tokunaga etal., "Influence of Chronic Intake of New Sweetener Fructooligosaccharide(Neosugar) on growth and Gastrointestinal Function of the Rat", JOURNALOF NUTRITIONAL SCIENCE AND VITAMINOLOGY, Vol. 32, pages 111-121 (1986).

As an indirect source of SCFAs, dietary fiber and indigestibleoligosaccharides (indigestable carbohydrate) can elicit certainmetabolic benefits. Total parenteral nutrition (TPN) or theadministration of a fiber free liquid diet leads to reduced colonic cellproliferation and atrophy. Janne et al., "Colonic Mucosal AtrophyInduced by a Liquid Elemental Diet in Rats", DIGESTIVE DISEASES, Vol.22, No. 9, pages 808-812 (1977); Morin et al., "Small Intestinal andColonic Changes Induced by a Chemically Defined Diet", DIGESTIVEDISEASES AND SCIENCES, Vol. 25, No. 2, pages 123-128 (1980); Sircar etal., "Effect of Synthetic Diets on Gastrointestinal Mucosal DNASynthesis in Rats", AMERICAN JOURNAL OF PHYSIOLOGY, Vol. 244, pagesG327-G335 (1983); Ryan et al., "Effects of Various Diets on ColonicGrowth in Rats", GASTROENTEROLOGY, Vol. 77, pages 658-663 (1979); Stormeet al., "The Effects of a Liquid Elemental Diet on Cell Proliferation inthe Colon of rats", CELL AND TISSUE RESEARCH, Vol. 216, pages 221-225(1981). Such atrophy could be prevented with the use of indigestiblecarbohydrate. Indigestible carbohydrate, through the production of SCFAsduring their fermentation, can stimulate colonic epithelial cellproliferation. Goodlad et al., "Proliferative Effects of Fibre on theIntestinal Epithelium", GUT, Vol. 28 pages 221-226 (1987); Kripke etal., "Stimulation of Intestinal Mucosal Growth with IntracolonicInfusion of Short-Chain fatty Acids", JOURNAL OF PARENTERAL AND ENTERALNUTRITION, Vol. 13, pages 109-116 (1989); Scheppach et al., "Effect ofShort-chain Fatty Acids on the Human Colonic Mucosa In Vitro", JOURNALOF PARENTERAL AND ENTERAL NUTRITION, Vol. 16, No. 1, pages 43-48 (1992);Sakata., "Stimulatory Effect of Short-chain Fatty Acids on EpithelialCell Proliferation in the Rat Intestine: A Possible Explanantion forTrophic Effects of Fermentable Fibre, Gut Microbes and Luminal TrophicFactors", BRITISH JOURNAL OF NUTRITION, Vol. 58, pages 95-103 (1987);Thomas et al., "Effect of enteral Feeding on Intestinal EpithelialProliferation and fecal Bile Acid Profiles in the Rat", JOURNAL OFPARENTERAL AND ENTERAL NUTRITION, Vol. 17, No. 3, pages 210-213 (1993).A recent animal study also has demonstrated the benefit of anindigestible carbohydrate in the treatment of experimental colitis.Rolandelli et al., "Comparison of Parenteral Nutrition and EnteralFeeding with Pectin in Experimental Colitis in the Rat", AMERICANJOURNAL OF CLINICAL NUTRITION, Vol. 47, pages 15-21 (1988).Specifically, the degree of bowel injury in experimental colitis wasdecreased when rats were fed an enteral diet supplemented with pectin,which is a dietary fiber. Improvements in outcome may have been due tothe SCFAs produced during the fermentation of pectin.

EXPERIMENT 1

In the first experiment the objective was to determine short chain fattyacid production from a variety of indigestible oligosaccharides duringfermentation with mixed human fecal microbiota. Several indigestibleoligosaccharides were tested including FOS, Raftilose® and XOS. FOS is afructooligosaccharide produced on a commercial scale by fermentinggranulated sucrose in water with a pure strain of Aspergillus niger. Theorganism produces a fructosyltransferase enzyme which links additionalfructose units onto the fructose end of sucrose molecules to produce1-kestose (GF₂), nystose (GF₃) and 1^(F) -β-fructo-furanosylnystose(GF₄). Raftilose® is a fructooligosaccharide produced via enzymatichydrolysis of inulin, which is marketed by Rhone-Poulenc (RaffinerieTirlemontoise SA). The hydrolysis results in a wide array ofoligosaccharides such as GF₂, GF₃ and GF₄ as well as oligosaccharidescontaining just fructose (F₃, F₄, F₅, etc.). XOS is axylooligosaccharide produced via enzymatic hydrolysis of xylan. Theprimary ingredients of XOS are xylobiose, xylotriose and xylotetrose.

The fermentation medium used in this first experiment is described inTable 3, and the anaerobic dilution solution used in this experiment isdescribed in Table 4.

                  TABLE 3                                                         ______________________________________                                        IN VITRO FERMENTATION MEDIUM COMPOSITION.sup.a                                INGREDIENT        AMOUNT (%)                                                  ______________________________________                                        Substrate (w/v)   1.0                                                         Salts A.sup.b (v/v)                                                                             33.0                                                        Salts B.sup.c (v/v)                                                                             33.0                                                        Salts SL6.sup.d (v/v)                                                                           1.0                                                         Vitamin mix.sup.e (v/v)                                                                         2.0                                                         Hemin Solution.sup.f (v/v)                                                                      0.25                                                        Resazurin solution.sup.g                                                                        0.10                                                        (v/v)                                                                         Yeast extract (w/v)                                                                             0.05                                                        Trypticase (w/v)  0.05                                                        Na.sub.2 CO.sub.3 (w/v)                                                                         0.40                                                        cysteine HCl H.sub.2 O (w/v)                                                                    0.05                                                        SCFA mix.sup.h (v/v)                                                                            0.04                                                        d H.sub.2 O (v/v) 31.88                                                       ______________________________________                                         .sup.a Media will be prepared as follows. All ingredients except              substrate, vitamin mix, Na.sub.2 CO.sub.3, cysteine HCl and SCFA mix will     be dissolved via boiling and then cooled to <50° C. through            bubbling with CO.sub.2. Na.sub.2 CO.sub.3 and the SCFA mix will then be       added and bubbled with CO.sub.2 until the solution is 30° C.           Substrate will then be added and the solution autoclaved for 15 min at        121° C. (15 psi). Cysteine HCl and the vitamin mix will be added t     the solution prior to dispensing.                                             .sup.b NaCl, 27.0 g; KH.sub.2 PO.sub.4, 13.5 g; CaCl.sub.2.H.sub.2 O, 0.8     g; MgCl.6H.sub.2 O, 0.6 g; MnCl.sub.2.4H.sub.2 O, 0.3 g;                      CoCl.sub.2.6H.sub.2 O, 0.3 g (NH.sub.4).sub.2 SO.sub.4, 27.0 g; d H.sub.2     O, 5.0 l.                                                                     .sup.c K.sub.2 HPO.sub.4, 13.5 g; d H.sub.2 O, 5.0 L.                         .sup.d EDTA disodium salts, 0.25 g; FeSO.sub.4 7H.sub.2 O, 0.1 g; d           H.sub.2 O, 450 ml; Minerals SL5 solution, 50 ml (ZnSO.sub.4.7H.sub.2 O,       0.04 g; MnCl.sub.2.4H.sub.2 O, 0.012; H.sub.3 PO.sub.4, 0.12 g;               CoCl.sub.2.6H.sub.2 O, 0.08 g; CuCl.sub.2 2H.sub.2 O, 0.004 g;                NiCl.sub.2.6H.sub.2 O, 0.008 g; Na.sub.2 MoO.sub.4.2H.sub.2 O, 0.012 g; d     H.sub.2 O, 400 ml).                                                           .sup.e ThiamineHCl, 0.05 g pantothenic acid, 0.05 g; niacin, 0.05 g;          pyridoxine, 0.05 g; riboflavin, 0.05 g; folic acid, 1.25 mg; biotin 1.25      mg; PABA, 2.5 mg; Vitamin B.sub. 12, 0.125 mg, d H.sub.2 O, 495 ml;           Vitamin K.sub.1 solution, 5.0 ml (vitamin K.sub.1, 125 μl, 95% ethanol     25.0 ml).                                                                     .sup.f hemin, 50 mg; 1 N NaOH, 1 ml; d H.sub.2 O, 99 ml.                      .sup.g See Table 4                                                            .sup.h Nvaleric acid, 0.2 ml; isovaleric acid, 0.2 ml; isobutyric acid,       0.2 ml; DLα-methylbutyric acid, 0.2 ml.                            

                  TABLE 4                                                         ______________________________________                                        ANAEROBIC DILUTION SOLUTION.sup.a                                             (1 Liter)                                                                     INGREDIENT          AMOUNT                                                    ______________________________________                                        Mineral solution 1.sup.b                                                                          37.5       ml                                             Mineral solution 2.sup.c                                                                          37.5       ml                                             Resazurin solution (.1% w/v).sup.d                                                                1.0        ml                                             NaHCO.sub.3         6.37       g                                              d H.sub.2 O (sonicated)                                                                           924.0      ml                                             cysteine HCl.H.sub.2 O                                                                            0.5        g                                              ______________________________________                                         .sup.a Mix minerals 1 and 2, resazurin and water, saturate with carbon        dioxide, and add NaHCO.sub.3 and autoclave. Add 0.5 g of cysteine HCl to      cooled solution.                                                              .sup.b K.sub.2 HPO.sub.4, 0.6 g; Na Citrate.2H.sub.2 O, 0.2 g; d H.sub.2      O, 100 ml.                                                                    .sup.c NaCl, 1.2 g; (NH.sub.4)SO.sub.4, 1.2 g; KH.sub.2 PO.sub.4, 0.6 g;      CaCl.sub.2, 0.12 g; MgSO.sub.4.7H.sub.2 O, 0.25 g; Na Citrate.2H.sub.2 O,     2 g; d H.sub.2 O 100 ml; (dissolve salts in H.sub.2 O in above order).        .sup.d Resazurin, 0.05 g; d H.sub.2 O, 50 ml.                            

The indigestible oligosaccharides were fermented in vitro for 3, 6, 12,and 24 hours with mixed human fecal microbiota. McBurney et al., "Effectof Human Faecal Inoculum on In Vitro Fermentation Variables", BRITISHJOURNAL OF NUTRITION, Vol.58, pages 233-243, (1987). Fermentations wererepeated with 3 donors because this is the minimal number required tomake accurate extrapolations to the general population. McBurney et al.,"Effect of Human Faecal Donor on In Vitro Fermentation Variables",SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, Vol. 24, pages 359-367,(1989). Briefly, a 0.115 g sample (dry weight) was weighed into a balchtube. Approximately 24 hours before the start of the incubation 10 mL ofthe fermentation medium described in Table 3 was added to the samples sothat the samples were hydrated when the inoculum was added. The redoxpotential of the contents of the tubes was reduced, the tubes werecapped with one-way valves and stored overnight in the refrigerator.,One to two hours before inoculation, the tubes were placed in a 37° C.water bath.

Fresh human feces was collected from three healthy individuals. Eachfecal sample was collected into a plastic bag. Air was expressed fromthe bag and an aliquot taken and mixed (blended under CO₂) with theanaerobic dilution solution described in Table 4 (40 g feces/360 mLanaerobic dilution solution; 1×10). The solution was filtered through 2layers of cheese cloth and the filtrate served as inoculum. Onemilliliter of this inoculum was injected into each tube. Tubes wereswirled at regular intervals. The fermentation was terminated at theappropriate time point (h) by opening the tubes and adding 2 mL 25%m-phosphoric acid.

Analysis of acetate, propionate and butyrate was conducted according toMerchen et al., "Effect of Intake and Forage Level on Ruminal andTurnover Rates, Bacterial Protein Synthesis and Duodenal Amino AcidFlows in Sheep", JOURNAL OF ANIMAL SCIENCE, Vol. 62, pages 216-225(1986). Briefly, an aliquot from the balch tube was acidified with 6NHCl and centrifuged at 31,000 ×g for 20 minutes Concentrations ofacetate, propionate and butyrate were determined in the supernatantusing a Hewlett-Packard 5890A gas chromatograph and a column (180 cm×4mm id) packed with 20% Tween 80-2% H₃ PO₄ on 60 to 80 mesh Chromosorb W(Supelco Inc, Bellefonte, Pa., U.S.A.). Nitrogen was used as a carriergas with a flow rate of 70 mL/minutes. Oven temperature was 120° C. anddetector and injector temperatures were 200° C. Lactate was determinedcolorimetrically using a method described in Barker et al., "TheColorimetric Determination of Lactic Acid in Biological Material",JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 138, page 535, (1941).

The experiment was analyzed as a randomized complete block with fecaldonor serving as the block. Treatments, which were arranged factorially,included substrate and length of fermentation. All analyses wereperformed using the General Linear Models procedure of StatisticalAnalysis Systems (SAS). Least significant difference (LSD) values forseparating treatment means at P<0.05 was 2.83 times the standard errorof the mean (SEM).

SCFA production (acetate, propionate, butyrate and lactate) during invitro fermentation of the oligosaccharides is presented in Table 3. Fourtime points were studied and include 3, 6, 12 and 24 hours. Retentiontime in the large bowel of humans will dictate the length offermentation in vivo. In cases where retention time is great, the extentof substrate fermentability will be a factor which most influences SCFAproduction. If retention time is short, the rate of substratefermentation becomes more important. Since retention times can differsignificantly in an in vivo situation it is necessary to monitorsubstrate degradation over time in vitro in order that comparisons canbe made.

Fermentation of all oligosaccharides was rapid, essentially beingcomplete by 6 hours for the fructooligosaccharides (FOS and Raftilose)and by 12 hours for XOS. The results are presented in Table 5. It isrecommended that the 6 hour and 12 hour values be used to estimate thecomposition of the end-products for the fructooligosaccharides and theXOS, respectively, even though retention times in the large bowel can beconsiderably longer. At later time points it becomes apparent thatlactate is being converted to propionate and acetate to butyrate.Interconversion in a closed in vitro system can be a problem withrapidly fermented substrates. It does not reflect the true state of thelarge bowel where the fatty acids are continually absorbed.

                                      TABLE 5                                     __________________________________________________________________________    SHORT CHAIN FATTY ACID                                                        PRODUCTION DURING 3, 6, 12 AND 24 H in vitro                                  FERMENTATION OF VARIOUS OLIGOSACCHARIDES                                                  SHORT CHAIN FATTY ACID.sup.a                                                                      Total                                         SUBSTRATE                                                                             HOUR                                                                              Acetate                                                                            Propionate                                                                          Butyrate                                                                           Lactate                                                                           SCFA.sup.b                                    __________________________________________________________________________    FOS     3   1.49 .20   .23  .45 2.37                                                  6   3.61 .54   .87  1.19                                                                              6.21                                                  12  3.67 1.01  1.64 .54 6.86                                                  24  3.20 1.09  2.09 .01 6.39                                          Raftilose ®                                                                       3   1.42 .20   .27  .47 2.36                                                  6   3.49 .53   .92  1.28                                                                              6.22                                                  12  3.68 .98   1.70 .59 6.95                                                  24  3.09 1.05  2.1  .01 6.30                                          XOS     3   1.21 .15   .13  .14 1.63                                                  6   4.12 .58   .58  .47 5.75                                                  12  5.90 .97   1.1  .74 8.72                                                  24  5.53 .96   1.5  .05 8.10                                          Statistics                                                                            SEM .13  .08   .08  .08                                                       LSD.sup.c                                                                         .37  .23   .23  .23                                               __________________________________________________________________________     .sup.a Calculated as (mmol fatty acid in incubation tube minus mmol fatty     acid in blank tube) divided by original substrate dry matter (DM) and         expressed as mmol/g substrate DM.                                             .sup.b Sum of acetate + propionate + butyrate + lactate and expressed as      mmol/g substrate DM.                                                          .sup.c Differences between mean values within a column greater than the       specified LSD are significandy different P < .05.                        

As is typically found with in vitro fermentations using human fecalinoculum or in analysis of fecal samples, acetate was the short chainfatty acid found in the highest concentration. Titgemeyer et al.,"Fermentability of Various Fiber Sources by Human Fecal Bacteria InVitro", AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol. 53, Pages1418-1424, (1991). Baldwin., "Energy Metabolism in Anaerobes", AMERICANJOURNAL OF CLINICAL NUTRITION, Vol. 23, No. 11, pages 1508-1513, (1970).determined that acetate, propionate and butyrate account for 83% of theSCFAs produced during anaerobic fermentation by large bowel microflora,and the remaining SCFAs are distributed among isovaleric, isobutyric,valeric, lactic, formic and succinic acids. In this study, aconsiderable amount of lactate was found, particularly duringfermentation with FOS and Raftilose. It has been documented that theoligosaccharides used in this study serve as an energy source forBifidobacteria and that there consumption will lead to the selectivegrowth of this organism in the GI tract. Okazaki et al., "Effects ofXylooligosaccharides on the Growth of Bifidobacteria", BIFIDOBACTERIAMICROFLORA, Vol. 9, No. 2, page 77, (1990); Mitsuoka et al., "Effects ofFructo-oligosaccharide on Intestinal Microflora", DIE NAHRUNG, Vol. 31,pages 427-436, (1987). The primary end products produced byBifidobacteria during fermentation are acetate and lactate. Miller etal., "Fermentations by Saccharolytic Intestinal Bacteria", AMERICANJOURNAL OF CLINICAL NUTRITION, Vol. 32, pages 164-172, (1979). The factthat these oligosaccharides serve as an energy source for theBifidobacteria could explain the elevated levels of lactate found inthis study.

Total short-chain fatty acid production was greater for thexylooligosaccharides (XOS) compared to the fructooligosaccharides (FOSand Raftilose). The primary factor effecting the quantity of SCFAsproduced during fermentation is the fermentability of the substrate. Itis assumed that the oligosaccharides are completely fermented in thissystem. However, the yield of SCFAs (mol) from a substrate is dependentnot only on the weight of the substrate fermented but also on theaverage molecular weight of the oligosaccharide component sugars. Onecan assume that the fermentation of one monosaccharide molecule canresult in either two acetate, two propionate, two lactate or onemolecule of butyrate. The molecular weight of the components of thefructooligosaccharides (glucose and fructose, 180) is greater than themolecular weight of xylose (150) which is the monomeric component ofXOS. Subsequently, on an equivalent weight basis, there are more molesof monosaccharide molecules with the xylooligosaccharide compared to thefructooligosaccharide. This would explain the greater production of SCFAwith the XOS compared to the fructooligosaccharides. Lastly, thequantity and profile of SCFAs produced was virtually identical betweenthe two fructooligosaccharides (Raftilose and FOS). While thesefructooligosaccharides differ to some extent in their chemicalcomposition, it is apparent that they are metabolized similarly in thisin vitro fermentation system.

In Experiment 1, the in vitro fermentability of three indigestibleoligosaccharides was evaluated. Fermentation of the indigestibleoligosaccharides was rapid and essentially complete by 12 hours. Suchcompounds may serve as an indirect energy source, particularly for thelarge bowel. By serving as an energy source the oligosaccharides may beuseful in preventing large bowel atrophy associated with the feeding ofsemi-elemental and elemental diets. Through the production of SCFAs, theindigestible oligosaccharides also may be useful in the treatment ofinflammatory bowel disease (ulcerative colitis). Also, considering theirability to serve as energy substrates for the anaerobic flora of thelarge bowel, particularly the Bifidobacteria, these compounds may beuseful in promoting the restoration of normal flora following antibiotictherapy or maintaining a normal flora in patients consuming enteraldiets. This may enhance colonization resistance to pathogens such as C.difficile. It is believed to be an important feature of the nutritionalproduct of the present invention that it contains a source ofindigestible carbohydrate which is metabolized to SCFAs bymicroorganisms present in the human colon and which comprise at leastone material selected from the group consisting of dietary fibers andindigestible oligosaccharides.

It is commonplace in western cultures for the predominant sources oflipids in the diet to be vegetable sources, such as corn or sunflowers,which provide relatively high amounts of linoleic acid (18:2n6).Linoleic acid can be metabolized to arachidonic acid (20:4n6) and henceto dienoic eicosanoids, such as prostaglandin E₂ (PGE₂), thromboxane A₂(TxA₂), and leukotriene B₄ (LTB₄). On the other hand, the predominantpolyunsaturated fatty acids present in fish oils are eicosapentaenoicacid (20:5n3) and docosahexaenoic acid (22:6n3). Eicosapentaenoic acid(20:5n3), which is not present in vegetable oils, has been shown to bemetabolized to a family of trienoic eicosanoids, for example,prostaglandin E₃ (PGE₃), thromboxane A₃ (TxA₃) and also leukotriene B₅(LTB₅) which have biological properties that are subtly different fromthose of the arachidonic acid (20:4n6) metabolites.

Although the primary etiology of ulcerative colitis is unknown, growingevidence supports a pathogenetic role of arachidonic acid derivedinflammatory mediators in this disorder. Eicosanoid formation isincreased in specimens from human diseased tissues. Sharon et al., "Roleof Prostaglandins in Ulcerative Colitis. Enhanced production duringActive Disease and Inhibition by Sulfasalazine", GASTROENTEROLOGY, Vol.75, No. 4, pages 638-640 (1978); Ligumsky et al., "Enhanced ThromboxaneA₂ and Prostacylcin Production by Cultured Rectal Mucosa in UlcerativeColitis and its Inhibition by Steroids and Sulfasalazine",GASTROENTEROLOGY, Vol. 81, pages 444-449 (1981); Sharon et al.,"Enhanced Synthesis of Leukotrine B₄ by Colonic Mucosa in InflammatoryBowel Disease" GASTROENTEROLOGY, Vol 86, pages 453-460 (1984). Luminaleicosanoid release measured in vivo in patients with active ulcerativerectocolitis is enhanced. Lauristen et al., "In Vivo Effects of OrallyAdministered Prednisolone on Prostaglandin and Leukotriene Production inUlcerative Colitis", GUT, Vol. 28 pages 1095-1099 (1987); Lauritsen etal., "In Vivo Profiles of Eicosanoids in Ulcerative Colitis, Crohn'sColitis and Clostridium Difficile Colitis", GASTROENTEROLOGY, Vol. 95,pages 11-17 (1988). Furthermore, animal and clinical work from a numberof laboratories suggests that elevated levels of leukotriene B₄,thromboxane and platelet activating factor participate in thedevelopment of chronic lesions. Seidman, "Nutritional Management ofInflammatory Bowel Disease", GASTROENTEROLOGY CLINICS OF NORTH AMERICA,Vol. 17, No. 1, pages 129-155 (1989); Dudrick et al., "NutritionalManagement of Inflammatory Bowel Disease", SURGICAL CLINICS OF NORTHAMERICA, Vol. 71, No. 3, pages 609-623 (1991); Teahon et al., "The Roleof Enteral and Parenteral Nutrition in Crohn's Disease and UlcerativeColitis", PROGRESS IN INFLAMMATION BOWEL DISEASE, Vol. 12, No. 2, pages1-4 (1991); Vilaseca et al., "Participation of Thromboxane and OtherEicosanoid Synthesis in the Course of Experimental InflammatoryColitis", GASTROENTEROLOGY, Vol. 98, pages 269-277 (1990).

Local eicosanoid generation by the gastrointestinal mucosa is modulatedby intraluminal, neural and hormonal factors. Among the intraluminalfactors, the diet might have a significant relevance in the regulationof mucosal eicosanoid biosynthesis, since the dietary intake ofprecursor fatty acids could directly influence the rate and pattern ofeicosanoid generation. Within the gastrointestinal tract, prostaglandinsderived from arachidonic acid have potent pro-inflammatory actions andcan alter motility, fluid secretion and electrolyte transport. Donowitz,"Arachidonic Acid Metabolites and Their Role in Inflammatory BowelDisease. An Update Requiring Addition of a Pathway", GASTROENTEROLOGY,Vol. 88, pages 580-587 (1985). In contrast, the lipoxygenase metabolites(leukotrienes) stimulate locomotion, superoxide production, lysosomalenzyme release in leukocytes, and colonic chloride secretion. Musch etal., "Stimulation of Colonic Secretion by Lipoxygenase Metabolites ofArachidonic Acid", SCIENCE (Washington, DC), Vol. 17, pages 1255-1256(1982); Palmer et al., "Chemokinetic Activity of Arachidonic AcidLipoxygenase Products on Leukocytes from Different Species",PROSTAGLANDINS, Vol. 20, No. 2, pages 411-448 (1980); Stenson et al.,"Monohydroxyeicosatetraenoic Acids (HETE's) Induce Degranulation ofHuman Neutrophils", JOURNAL OF IMMUNOLOGY., Vol. 124, No. 5, pages2100-2104 (1980). These products of arachidonate metabolism are thuspotential mediators of ulcerative colitis and may account foralterations in intestinal fluid and electrolyte secretion.

Recent evidence that the regular intake of n-3 fatty acids from fish oilinhibits neutrophil and monocyte functions suggests that n-3 fatty acidshave antiinflammatory properties. Beneficial effects of marine lipidshave been shown in animal models of inflammatory bowel disease. Empey etal., "Fish Oil-Enriched Diet is Mucosal Protective Against AceticAcid-Induced Colitis in Rats", CANADIAN JOURNAL OF PHYSIOLOGY ANDPHARMACOLOGY, Vol. 69, pages 480-487 (1991); Vilaseca et al., "DietaryFish Oil Reduces Progression of Chronic Inflammatory Lesions in a RatModel of Granulomatous Colitis", GUT, Vol. 31, page 539 (1990). Inpreliminary therapeutic trials, diet supplementation with fish oil hasled to symptomatic improvement of patients with ulcerative colitis, andreduced ethanol-induced damage in human duodenal mucosa. Schepp et al.,"Fish Oil Reduces Ethanol-Induced Damage of the Duodenal Mucosa inHumans", EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Vol. 21, pages230-237 (1991); Lorenz et al., "Supplementation with n-3 Fatty Acidsfrom Fish Oil in Chronic Inflammatory Bowel Disease", JOURNAL OFINTERNAL MEDICINE SUPPLEMENT, Vol. 225, pages 225-232 (1989); Hillier etal., "Incorporation of Fatty Acids from Fish Oil and Olive Oil intoColonic Mucosal Lipids and Effects Upon Eicosanoid Synthesis inInflammatory Bowel Disease", GUT, Vol. 32, pages 1151-1155 (1991);Saloman et al., "Treatment of Ulcerative Colitis with Fish OilN-3-w-Fatty Acid: An Open Trial", JOURNAL OF CLINICAL GASTROENTEROLOGY,Vol. 12, No. 2, pages 157-161 (1990).

When abundant n-3 fatty acids in the form of fish oil are included inthe diet, eicosapentaenoic (EPA) and docosahexaenoic acid may inhibitthe synthesis of arachidonic acid from linoleic acid, reduce plasmalevels of arachidonic acid by competing for incorporation into membranephosphol ipids, and compete with arachidonic acid as a substrate forcyclooxygenase, and to a greater extent, lipoxygenase metabolism.Production of the 2-series prostaglandins (PGE₂, TXB₂), and the 4-seriesleukotrienes (LTB₄, LTC₄) are thus diminished, and the less biologicallyactive 3-series prostaglandins (PGE₃, TXB₃) and 5-series leukotrienes(LTB₅, LTC₅) are formed. It is through these mechanisms that dietaryfish oils are thought to manifest their anti inflammatory effects.Fretland et al., "Eicosanoids and Inflammatory Bowel Disease: Regulationand Prospects for Therapy", PROSTAGLANDINS LEUKOTRINES AND ESSENTIALFATTY ACIDS, Vol. 41, pages 215-233, at pages 224-225 (1990) relate thatin a small uncontrolled pilot study of ulcerative colitis patients givenfish oil capsules (Max EPA) containing 3-4 grams of EPA daily for twelveweeks showed significant improvement in symptoms and histologicalappearance of the rectal mucosa by the end of the treatment period.Neutrophil LTB₄ levels fell significantly during the treatment. The MaxEPA also contained some vitamin E, a compound with antioxidant andantiinflammatory properties, which could conceivably have accounted forsome of the therapeutic effect. Dietary vitamin E supplementationhowever, was shown not to promote changes in eicosanoid levels obtainedfrom rectal dialysate fluid of active ulcerative colitis patients in aseparate study.

EXPERIMENT 2

A major limitation in investigating the pathogenic mechanismsresponsible for the mucosal injury observed during chronic inflammationof the intestine and colon has been the relative paucity of relevantanimal models. Two models of colitis produced in rats that have receivedmuch attention over the past few years are the acetic acid andtrinitrobenzene sulfonic acid (TNBS) models. The mechanism by whichacetic acid produces the diffuse colitis is thought to involvenonspecific, acid induced injury to the colonic mucosa that is followedby an acute inflammatory response. Apparently the protonated form of theacid is required to induce the colitis since neither HCl (pH 2.3) norsodium acetate (pH 7.0) is effective in eliciting the inflammatoryresponse. However, there is some evidence to suggest that acetic acidmay promote other pathophysiological events (e.g. fluid and electrolytesecretion) using noncytotoxic concentrations of the acid.

Recent studies have demonstrated that the intrarectal administration ofthe hapten, TNBS, in the presence of a mucosal barrier breaker such asethanol, produces an acute and possibly chronic colitis in unsensitizedrats. The mechanism(s) by which buffered or unbuffered TNBS in thepresence of ethanol initiates inflammation in unsensitized animals isunclear; however, it has been suggested to involve macrophage-mediatedrecognition and lysis of TNBS-modified autologous cells within themucosa. However, more recent evidence suggests more complicatedmechanisms. For example, the barrier breaker, ethanol, is an extremelypotent pro-inflammatory solvent alone. Furthermore, it has beendemonstrated that TNBS is metabolized by certain colonic enzymes andsubstrates to yield both pro-inflammatory and cytotoxic oxidants thatcould initiate colonic inflammation. Grisham et al., "Metabolism ofTrinitrobenzene Sulfonic Acid by the Rat Colon Produces Reactive OxygenSpecies", GASTROENTEROLOGY, Vol. 101, pages 540-547 (1991). A recentstudy directly compared the acetic acid and the TNBS (+ETOH) models ofcolitis and found that either model may be useful to study those eventsthat occur at the time of inflammation (e.g. arachidonate metabolism,granulocyte infiltration and metabolism, etc.) or during repair.However, the use of these models of colitis may have significantlimitations in understanding those immunological events that initiatethe acute and chronic inflammatory episodes. For example, theinflammation and tissue injury observed in human inflammatory boweldisease is most probably a result of inappropriate immunologicalactivation (e.g. autoimmune, infectious agent, etc.) whereas theinflammation induced by the intrarectal application of acetic acid,ethanol or ethanol plus TNBS is a response to extensive mucosal injury.Thus, the mechanisms by which inflammation (and mucosal injury) areachieved in the human disease may be very different than those in theexperimental models.

For these reasons, a model of acute and chronic distal colitis in ratswas developed based upon a previously published method in which purifiedbacterial cell wall polymers (derived from Group A streptococci) areinjected intramurally into the distal colon of genetically-susceptiblerats. Sartor et al., "Granulomatous Entercolitis Induced by PurifiedBacterial Cell Wall Fragments", GASTROENTEROLOGY, Vol. 89, pages 587-595(1985). This model produces an acute and chronic inflammationcharacterized by the infiltration of large numbers of inflammatorycells, enhanced mucosal permeability, interstitial fibrosis, and mucosalthickening as well as the extraintestinal manifestations of arthritis,hepatic and splenic granulomas. Unlike most models of colitis, theinflammation induced in this model promoting mucosal and submucosalinjury rather than the injury causing the inflammation.

In Experiment 2 the objectives were: (a) to determine whether this modelof colitis responds to sulfasalazine (SAZ) and (b) to assess the effectsof specially formulated enteral diets on the injury and inflammationobserved in the colon, liver and spleen.

Female Lewis rats (150-175 g) were maintained in a controlledtemperature and light-dark cycle (12 hours:12 hours) and housed inwire-mesh bottomed cages and given water and standard laboratory ratchow ad libitum. A total of 48 rats were divided into 6 groups of 8 ratseach consisting of a sham (rats receiving Diet #1 {base diet} with nopeptidoglycan/polysaccharide {PG/PS}injection) control group, a chowgroup and 4 groups of rats placed on 4 different polymeric diets (Table6). Rats received either chow or polymeric diets (320 kcals/kg/day or 60mL of liquid diet/day) for 7 days preceding induction of colitis. Distalcolitis was induced by a modification of the method of Sartor et al inwhich multiple sites (8-9) along the distal colon were injectedintramurally with 60 ul/site to deliver a dose ofpeptidoglycan/polysaccharide (PG/PS) of 12.5 ug/g body weight. Thealbumin (sham) control group received the same number of injections ofhuman serum albumin into the distal colon.

                                      TABLE 6                                     __________________________________________________________________________    INFLAMMATORY BOWEL DISEASE FORMULATIONS                                       (Formulations expressed as percentage of calories)                            Diet.sup.a                                                                         Lipid.sup.b                                                                             Carbohydrate                                                                            Protein   Fiber.sup.c                                __________________________________________________________________________    Control                                                                            18.0%     61.0%     21.0%     None                                            14.7%                                                                             corn oil                                                                            42.7%                                                                             hydrolyzed                                                                          16.0%                                                                             caseinates                                            2.7%                                                                              MCT       cornstarch                                                                          5.0%                                                                              hydrolyzed                                            0.6%                                                                              soy lecithin                                                                        18.3%                                                                             sucrose   soy.sup.d                                        Fish oil                                                                           18.0%     61.0%     21.0%     None                                            1.6%                                                                              canola oil                                                                          42.7%                                                                             hydrolyzed                                                                          16.0%                                                                             caseinates                                            11.7%                                                                             fish oil  cornstarch                                                                          5.0%                                                                              hydrolyzed                                            2.7%                                                                              MCT   18.3%                                                                             sucrose   soy                                                   1.4%                                                                              soybean oil                                                               0.6%                                                                              soy lecithin                                                         FOS  18.0%     61.0%     21.0%     4.5 g                                           14.7%                                                                             corn oil                                                                            42.7%                                                                             hydrolyzed                                                                          16.0%                                                                             caseinates                                                                          2.25 g                                                                            FOS                                         2.7%                                                                              MCT       cornstarch                                                                          5.0%                                                                              hydrolyzed                                                                          2.25 g                                                                            gum                                         0.6%                                                                              soy lecithin                                                                        18.3%                                                                             sucrose   soy       arabic                                 XOS  18.0%     61.0%     21.0%     4.5 g                                           14.7%                                                                             corn oil                                                                            42.7%                                                                             hydrolyzed                                                                          16.0%                                                                             caseinates                                                                          2.25 g                                                                            XOS                                         2.7%                                                                              MCT       cornstarch                                                                          5.0%                                                                              hydrolyzed                                                                          2.25 g                                                                            gum                                         0.6%                                                                              soy lecithin                                                                        18.3%                                                                             sucrose   soy       arabic                                 __________________________________________________________________________     .sup.a The caloric density of all diets is 1.2 kcal/mL. The nutrient base     is 1250 kcal. As used herein "nutrient base" means the amount of calories     of a product that must be consumed to provide 100% of the US RDA of           vitamins and minerals for humans.                                             .sup.b Mochida concentrated fish oil (28% EPA:12% DHA).                       .sup.c Fiber is expressed as g/8 fl oz. FOS (Fructooligosaccharide, Golde     Technologies Inc.), gum arabic (Nutriloid arabic, TIC Gums), XOS              (Xylooligosaccharide, Suntory, Inc.).                                         .sup.d Soy protein hydrolyzate PP750 (slightly hydrolyzed).              

Total dietary intake and body weights of the control and liquid dietgroups were recorded for each 24 hour period during the course of the 4week experiment (1 week prior to the induction of colitis and 3 weeksfollowing PG/PS or albumin injection). To assess the effects of SAZ inthis model, female Lewis rats were orally administered SAZ immediatelyfollowing the induction of colitis. Rats were given chow ad libitum forthe duration of the four week study period. Similar measurements wereassessed as described below.

All rats receiving nutritional therapy or SAZ were euthanized with anoverdose of pentobarbital and the distal colon and cecum were excisedand opened longitudinally. The length and weight of the perfused segmentwere recorded and the tissue divided longitudinally into three stripsfor wet-to-dry ratios, histology and myeloperoxidase (MPO)determinations. Colonic MPO activity was determined in which 3,3' 5,5'tetramethylbenzidine was used as the electron donating substrate andhexadecyltrimethylammonium hydroxide was used as the detergent. Spleenand liver weights were recorded. Circulating levels of nitrate andnitrite were also quantified using the Griess reagent and used asindices of immune system activation (i.e. activation of thereticuloendothelial system {nitric oxide synthase}). The studies wereanalyzed as a completely randomized design. Treatment differences wereseparated using an F-protected least significant difference (LSD)method. In both studies additional chow fed control animals that weretreated under laboratory conditions described above were incorporated inthe statistical analysis.

Effects of SAZ administration are presented in Table 7. It was foundthat oral administration of SAZ beginning immediately followinginduction of colitis significantly attenuated the increases in MPOactivity and tended to reduce colon weight compared to a chow fed groupreceiving PG/PS suggesting that this antiinflammatory agent inhibitsgranulocyte infiltration and fibrosis (we observed no significantincrease in wet to dry ratios in our inflamed bowel suggesting that theincreases in colon weight were due to collagen deposition). Histologicalinspection of the SAZ-treated tissue confirmed inhibition of leukocyteinfiltration and fibrosis. Previous studies have demonstrated that theinflammation induced by the intramural (subserosal) injection of PG/PSis primarily submucosal in nature and heterogeneous with respect toepithelial cell injury. For example histological inspection of thistissue reveals foci of modest epithelial injury surrounded by regions ofa completely intact epithelium.

In addition to its protective effects on the colon, SAZ treatmentresulted in liver and spleen weights significantly lower than the PG/PStreated animals and comparable to the sham control suggesting that SAZinhibits granuloma development and necrosis in these two organs. Atpresent, the mechanism by which SAZ protects these two organs remainsundefined. It may be that SAZ protects primarily the gut therebyinhibiting the emigration of noxious luminal antigens and bacterialproducts into the portal and systemic circulation where they may promotedistal organic inflammation. Alternatively, SAZ or one of its activemetabolites (e.g. 5-ASA) is present in the systemic circulation and mayexert direct antiinflammatory action on the various organ systems. Theantiinflammatory mechanism of SAZ remains the subject of active debate.There is a large body of experimental data to suggest that the activemoiety of SAZ is 5-ASA which is a potent antioxidant and is a modest5-lipoxygenase inhibitor. In addition, there is emerging evidence tosuggest that the parent diazo compound (ie., SAZ) may possesssignificant antiinflammatory activity.

Recent studies have suggested that nutritional supplementation in theform of enteral diets may prove useful as adjunctive or primary therapyfor patients with IBD. Indeed, recent reports suggest that n-3 fattyacids from fish oil as well as the SCFA produced during the fermentationof indigestible carbohydrates may attenuate some of the pathophysiologyassociated with active gut inflammation. Therefore, we ascertainedwhether three enteral diets, one supplemented with fish oil or twodifferent diets supplemented with two forms of indigestible carbohydratecould inhibit some of the inflammation observed in a model of chroniccolitis. The results are presented in FIGS. 1-5. FIG. 1 presents colonweights of animals following the various therapies (diets). FIG. 2presents MPO activity in colonic tissue of rats following the varioustherapies (diets). FIG. 3 presents liver weights of animals followingthe various therapies (diets). FIG. 4 presents spleen weights in theanimals following the various therapies (diets). FIG. 5 presents levelsin circulating plasma of nitrate and nitrite in animals following thevarious therapies (diets).

                                      TABLE 7                                     __________________________________________________________________________    EFFECTS OF SAZ                                                                                MPO                                                                   COLON WT                                                                              ACTIVITY Liver wt                                                                            Spleen wt                                      TREATMENT                                                                             (g dry wt/cm)                                                                         (units/cm colon)                                                                       (mg/g bw)                                                                           (mg/g bw)                                      __________________________________________________________________________    Sham Chow                                                                             .016 ± .003.sup.b                                                                  1.17 ± 2.5.sup.b                                                                    31.5 ± 3.1.sup.b                                                                 2.08 ± 1.1.sup.b                            PG/PS Chow                                                                            .029 ± .002.sup.a                                                                  8.49 ± 1.6.sup.a                                                                    45.0 ± 2.0.sup.a                                                                 6.95 ± .70.sup.a                            SAZ     .021 ± .004.sup.a,b                                                                1.40 ± 2.9.sup.b                                                                    34.0 ± 3.6.sup.b                                                                 2.92 ± 1.3.sup.b                            .sup.c P =                                                                            .0045   .0195    .0008 .0007                                          __________________________________________________________________________     .sup.a,b Least Square means with unlike superscript letters differ (P <       .05).                                                                         .sup.c Overall treatment effect.   Results indicate that all enteral diet     used in this study provided for a certain degree of antiinflammatory     activity. The addition of fish oil or indigestible oligosaccharides such     as FOS and XOS demonstrated greater antiinflammatory activity compared to     the base control or chow fed PG/PS rats. Antiinflammatory activity was     ascertained using colon, liver and spleen weights along with MPO activity.     Results show that the control diets as well as the fish oil and XOS diets     produced colon weights that were significantly lower than chow fed PG/PS     animals and comparable to the sham control (FIG. 1). While all enteral     diets tended to attenuate MPO activity compared to chow PG/PS fed animals,     only the fish oil and XOS diets were significantly lower than chow animals     (FIG. 2). Only the chow fed PG/PS rats resulted in an MPO activity that     was significantly greater than the sham control. Liver weights also were     affected by the diet (FIG. 3). The FOS and XOS diets resulted in liver     weights that were significantly lower than the chow fed PG/PS animals.     Only chow fed PG/PS rats had liver weights that were significantly     different than sham control animals. The fish oil, FOS and XOS diets     resulted in spleen weights that were comparable to the sham control (FIG.     4). The control and chow diets produced spleen weights that were greater     than the sham control. All enteral diets significantly attenuated the     increase in nitrate and nitrite compared to chow fed animals. These levels     were comparable to sham controls. This is of importance because it has     been proposed that the large increases in circulating levels of nitrate     and nitrite such as in the PG/PS chow fed group, arise from the production     of nitric oxide by extravasated polymorphonuclear cells, monocytes and     macrophages.

These results demonstrate that a complete enteral diet (control diet)given to rats for 28 days with distal colitis, reduced disease activityas indicated by the above indices of inflammation. Howeversupplementation with the bioactive ingredients, fish oil, FOS or XOS,showed additional antiinflammatory activity by significantly attenuatingthe colonic and extraintestinal inflammation associated with distalcolitis. In most instances, these indices of inflammation were similarto those indices in chow fed sham controls. The antiinflammatoryactivity of these diets was confirmed via histological inspectionshowing an inhibition of inflammation and maintenance of crypt cellintegrity.

An interesting aspect of the present study is that supplementation ofenteral diets with fish oil or indigestible oligosaccharides rendersthese diets similar in efficacy to a known antiinflammatory drug (SAZ)used to treat human IBD and which has been shown to be efficacious inthis model of inflammation. Although enteral diets may be considered asalternative primary therapy for chronic gut inflammation, it may be moreuseful to consider their use as adjunctive therapy to be used incombination with steroids and/or aminosalicylates.

It is believed to be an important feature of an enteral nutritionalproduct of the present invention that it contains an oil blend whichcomprises, by weight, a total of at least 25% of one or more oilsselected from the group of oils which contain eicosapentaenoic acid(20:5n3) and docosahexaenoic acid (22:6n3). The nutritional product ofthe invention may comprise the oil blend presented in Table 8. Otherfeatures of an oil blend useful in the practice of the present inventionare presented in Table 9 and 10.

                  TABLE 8                                                         ______________________________________                                        ULCERATIVE COLITIS PRODUCT OIL BLEND                                          (as % of total weight of oil blend)                                           OIL         TARGET    PREFERRED RANGE                                         ______________________________________                                        Canola Oil  9.3%       5.0%-40.0%                                             MCT         16.2%     10.0%-50.0%                                             Fish Oil    65.0%     25.0%-80.0%                                             Soybean Oil 5.5%       3.0%-30.0%                                             Soy Lecithin                                                                              4.0%      2.0%-6.0%                                               ______________________________________                                    

Other oils that could be used in this product are: Corn oil, saffloweroil, sunflower oil, high-oleic safflower oil, high-oleic sunflower oil,olive oil, borage oil, black current seed oil and evening primrose oil.

                  TABLE 9                                                         ______________________________________                                        FATTY ACID PROFILE OF NEW LIPID BLEND                                         (as % of total fatty acids by weight, by analysis)                            ______________________________________                                        Caproic (6:0)       0.53                                                      Capyrlic (8:0)      10.35                                                     Capric (10:0)       7.16                                                      Lauric (12:0)       0.29                                                      Myristic (14:0)     3.53                                                      Palmitic (16:0)     7.41                                                      Palmitoleic (16:1n7)                                                                              5.73                                                      Stearic (18:0)      1.39                                                      Oleic (18:1n9)      15.23                                                     Linoleic (18:2n6)   7.21                                                      Gamma-Linolenic (18:3n6)                                                                          0.21                                                      Alpha-linolenic (18:3n3)                                                                          2.21                                                      Stearidonic (18:4n3)                                                                              2.40                                                      Arachidic (20:0)    0.13                                                      Eicosenoic (20:1n9) 0.74                                                      Arachidonic (20:4n6)                                                                              0.87                                                      Eicosapentaenoic (20:5n3)                                                                         17.14                                                     Erucic (22:1n9)     0.17                                                      Docosapentaenoic (22:5n3)                                                                         2.08                                                      Docosahexaenoic (22:6n3)                                                                          7.73                                                      Nervonic (24:1n9)   0.14                                                      Others              7.35                                                      TOTAL               100.00                                                    ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        FATTY ACID LEVELS IN OIL BLEND                                                (percent of total fatty acids)                                                                                 MOST                                                              PREFERRED   PREFERRED                                    FATTY ACID TARGET    RANGE       RANGE                                        ______________________________________                                        Oleic acid 13.5%     11.5%-15.7% 12.1%-15.1%                                  (18:1n9)                                                                      Linoleic acid                                                                            7.8%      6.6%-9.0%   7.0%-8.6%                                    (18:2n6)                                                                      Alpha-Linolenic                                                                          1.8%      1.5%-2.1%   1.6%-2.0%                                    acid (18:3n3)                                                                 Eicosapentaenoic                                                                         17.8%     15.1%-20.5% 16.0%-19.6%                                  acid (20:5n3)                                                                 Docosahexaenoic                                                                          7.5%      6.3%-8.6%   6.7%-8.3%                                    acid (22:6n3)                                                                 n-6/n-3 ratio                                                                            0.32      0.25-4.0                                                 18:2n6/18:3n3                                                                            4.26       3.0-10.0                                                ______________________________________                                         *The n6 fatty acids which are used in determining the n6/n-3 ratio for th     product disclosed herein are: Linoleic (18:2n6), Eicosadienoic (20:2n6)       and Arachidonic (20:4n6). The n3 fatty acids which are used in determinin     the n6/n-3 ratio for the product disclosed herein are: Alphalinolenic         (18:3n3), Stearidonic (18:4n3), Eicosapentaenoic (20:5n3),                    Docosapentaenoic (22:5n3) and Docosahexaenoic (22:6n3).                  

While not intending to be bound by theory, the combination ofindigestible carbohydrate and specifically dietary fiber andindigestible oligosaccharides with fish oil may increase theincorporation of n-3 fatty acids into colonocytes. The incorporation ofn-3 fatty acids into colonocytes of persons consumingpolymeric/elemental diets devoid of indigestible fermentable material isslow. Hypoproliferation of colonocytes and atrophy is documented withelemental low residue diet feedings. Fermentable indigestibleoligosaccharide such as fructooligosaccharides can promote cellproliferation. Rate and extent of n-3 fatty acid incorporation intocolonocytes is dependent on exchange of plasma n-3 fatty acids and colonmucosal phospholipids and rate of colonic cell turnover. The maintenanceor promotion of cell proliferation due to the incorporation ofindigestible carbohydrate into a liquid diet containing n-3 fatty acidscould promote a rapid increase of n-3 fatty acids from fish oil intocolonic mucosal lipids compared to a liquid diet devoid of indigestiblecarbohydrate. The therapeutic benefit of increasing the incorporation ofn-3 fatty acids into colonic mucosal phospholipids is to (a) promote anantiinflammatory effect by modulating local eicosanoid generation by thegastrointestinal mucosa of ulcerative colitis patients and (b) promotethe rapid incorporation of n-3 fatty acids from fish oil in thegastrointestinal mucosa of ulcerative colitis patients which willdecrease the hyperimmune response resulting in reduced mucosalulceration and disease activity index.

A growing body of data indicates that oxygen derived free radicals suchas superoxide (O₂.sup..), hydrogen peroxide (H₂ O₂), and hydroxylradicals (OH.sup..) have a role in mediating intestinal damage ininflammatory bowel disease. The most probable source of these oxidantsare the phagocytic leukocytes since these cells are known to be presentin large numbers in the inflamed mucosa and have been shown to producesignificant amounts of reactive oxygen species in response to certaininflammatory stimuli. Grisham, "Role of Neutrophil--Derived Oxidants inthe Pathogenesis of Inflammatory Bowel Disease", PROGRESS ININFLAMMATORY BOWEL DISEASE, Vol. 12, No. 1, pages 6-8 (1991). Grisham etal., "Neutrophil-Mediated Mucosal Injury. Role of Reactive Metabolites",DIGESTIVE DISEASES AND SCIENCES, Vol. 33, No. 3, pages 6-15S (1988),have hypothesized that in ulcerative colitis, transientischemia-reperfusion episodes produce high levels of free radicalsresulting in mucosal ulceration.

Grisham et al., "Oxidant Mechanisms in the Human Colon", INFLAMMATION,Vol. 14, No. 6, pages 669-680 (1990) have determined that the normalcolon, particularly the mucosa, contains small amounts of antioxidantenzyme systems such as superoxide dismutase, catalase, and GSH peroxidesuggesting that the colon may be susceptible to oxidant-mediated damage.Data, however, on endogenous antioxidant proteins in the intestinalmucosa of patients with inflammatory bowel disease are lacking. The twomost important copper and zinc containing proteins with radicalscavenging potential are metallothionein and superoxide dismutase.Metallothionein is a metal binding protein whose function is theregulation of copper and zinc metabolism. Thornalley et al., "PossibleRole for Metallothionein in Protection Against Radiation--InducedOxidative Stress. Kinetics and Mechanism of its Reaction with Superoxideand Hydroxyl Radicals", BIOCHIMICA ET BIOPHYSICA ACTA, Vol. 827, pages36-44 (1985), were the first to note the high OH.sup.. scavengingpotentials of metallothionein. Since then it has been found to protectDNA molecules, cells in culture, and whole organisms against thedetrimental effects of several types of free radical generatingtreatments. Abel et al., "Inhibition of Hydroxyl-Radical-Generated DNADegradation by Metallothionein", TOXICOLOGY LETTERS, Vol. 47, pages191-196 (1989); Bakka et al., "Radioresistance in Cells with HighContent of Metallothionein", EXPERENTIA SUPPLEMENTUM, Vol. 38, pages381-383 (1982); Matsubara, "Alteration of Radiosensitivity inMetallothionein Induced Mice and a Possible Role of Zn-Cu-Thioneine inGSH--Peroxidase System", EXPERENTIA, Vol. 52, pages 603-613 (1987).Furthermore, Mulder et al., "Decrease in Two Intestinal Copper/ZincContaining Proteins with Antioxidant Function in Inflammatory BowelDisease", GUT, Vol. 32, pages 1146-1150 (1991), found that superoxidedismutase content was similar in control mucosa and non-inflamed mucosafrom patients with inflammatory bowel disease but was decreased ininflamed mucosa. Similar results were also shown with metallothionein.Overall, a decrease in endogenous intestinal protection against oxygenderived radicals in inflammatory bowel disease may contribute to thepathogenesis of the disease.

Considering the compromised antioxidation state of the large bowel ofinflammatory bowel disease patients, it would be beneficial to increaseintakes of vitamins which have antioxidant properties. Vitamins E, C andbeta-carotene are among the most important of these antioxidantvitamins, but the minerals manganese, copper, zinc and selenium are alsorequired for the functional status of the antioxidant enzymes,metallothionein and superoxide dismutase. In a preferred embodiment theenteral nutritional product of the present invention contains at leastone nutrient selected from the group consisting of beta-carotene,vitamin E, vitamin C, taurine and selenium.

An enteral nutritional product according to the present invention hasabout 18.5%-23.5% (most preferably about 21.0%) of total caloriesprovided by protein, about 59.0%-63.0% (most preferably about 61.0%) oftotal calories provided by carbohydrate, and about 16.0%-20.0% (mostpreferably about 18.0%) of total calories provided by fat. Preferablythe protein source contains at least one material selected from thegroup consisting of intact and hydrolyzed (regardless of degree ofhydrolysis) proteins of high biological value. "High biological value"is understood to mean a protein source which provides a full complementof amino acids to the body An enteral nutritional product according tothe present invention preferably contains about 20 g of indigestiblecarbohydrate per liter. The source of indigestible carbohydrate may beselected from gum arabic, soy polysaccharide, fructooligosaccharides,hydrolyzed inulin, xylooligosaccharides or any other suitable material.

Key features of the nutritional product are presented in Table 11, theamino acid profile of the product is presented in Table 12 and a morecomplete nutrient profile of the enteral nutritional product of thepresent invention is presented in Table 13.

                  TABLE 11                                                        ______________________________________                                        IMPORTANT NUTRIENT FEATURES OF PRODUCT                                        NUTRIENT  TARGET       PREFERRED RANGE                                        ______________________________________                                        Protein   21.0% (67.8 g/L)                                                                           18.5%-23.5%                                                                   (59.7 g/L-75.9 g/L)                                    Carbohydrate                                                                            61.0% (201.5 g/L)                                                                          59.0%-63.0%                                                                   (194.9 g/L-208.1 g/L)                                  Fat       18.0% (28.2 g/L)                                                                           16.0%-18.0%                                                                   (25.1 g/L-31.3 g/L)                                    Beta-carotene                                                                           5,000 ug/L   2,500 ug/L-6,500 ug/L                                  Vitamin E 300 IU/L     100 IU/L-450 IU/L                                      Vitamin C 650 mg/L     250 mg/L-850 mg/L                                      Taurine   275 mg/L     200 mg/L-350 mg/L                                      Indigestible                                                                            19.89 g/L    16.91 g/L-22.87 g/L                                    Carbohydrate                                                                  ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        AMINO ACID PROFILE OF PRODUCT                                                 (Per actual analysis, values normalized to 100%)                              AMINO ACID     g/100 g Protein                                                ______________________________________                                        Aspartic Acid  7.08                                                           Threonine      4.34                                                           Serine         5.68                                                           Glutamic Acid  20.58                                                          Proline        10.55                                                          Glycine        1.81                                                           Alanine        3.04                                                           Valine         5.90                                                           Methionine     2.78                                                           Isoleucine     4.77                                                           Leucine        9.08                                                           Tyrosine       4.79                                                           Phenylalanine  4.96                                                           Histidine      2.67                                                           Lysine         7.27                                                           Arginine       3.15                                                           Tryptophan     0.99                                                           Cystine        0.56                                                           ______________________________________                                    

                  TABLE 13                                                        ______________________________________                                        PREFERRED NUTRIENT PROFILE OF THE                                             ULCERATIVE COLITIS NUTRITIONAL PRODUCT                                                       TARGET      ACCEPTABLE                                         RANGE/QUANTITY QUANTITY/   QUANTITY/                                          NUTRIENT       LITER       LITER                                              ______________________________________                                        Protein, g     67.4        66-70                                              Fat, g         27.2        25-29                                              Carbohydrate, g                                                                              207         204-215                                            Total Dietary  10.7         9.1-12.3                                          Fiber, g                                                                      Indigestible   12.4        10.5-14.3                                          Oligosaccharide                                                               (FOS), g                                                                      Gum Arabic, g  9.1          7.7-10.5                                          Soy            1.6         1.4-1.8                                            Polysaccharide, g                                                             β-carotene, μg                                                                       5000        2500-6500                                          Vitamin A, IU  5500        4500-6500                                          Vitamin D, IU  800         675-950                                            Vitamin E, IU  300         100-450                                            Vitamin K.sub.1, μg                                                                       135         120-150                                            Vitamin C, mg  650         250-850                                            Folic Acid, μg                                                                            1900        1688-2150                                          Thiamine, mg   6.5         2.53-8.0                                           Riboflavin, mg 5           2.87-6.5                                           Vitamin B.sub.6, mg                                                                          5           3.38-6.5                                           Vitamin B.sub.12, μg                                                                      18          10.1-25.0                                          Niacin, mg     40          33.8-50.0                                          Choline, mg    525         506-900                                            Biotin, μg  750          506-1000                                          Pantothenic Acid,                                                                            24          16.9-30                                            mg                                                                            Sodium, mg     1500        1350-1650                                          Potassium, mg  2000        1800-2200                                          Chloride, mg   1519        1367-1671                                          Calcium, mg    1800        1477-1920                                          Phosphorous, mg                                                                              1250        1055-1372                                          Magnesium, mg  450         422-550                                            Iodine, μg  175         158-300                                            Copper, mg     2.61        2.25-3.0                                           Zinc, mg       29.2        25.3-35.0                                          Iron, mg       22.2        20.3-25.0                                          Selenium, μg                                                                              90          78.8-125                                           Chromium, μg                                                                              125         112.5-150                                          Molybendum, μg                                                                            206         168.8-250                                          Carnitine, mg  150         127-200                                            Taurine, mg    275         200-350                                            Kcal/mL        1.29        1.27-1.34                                          ______________________________________                                         *d-alpha-tocopheryl (all natural form) or dlalpha tocopherol acetate, or      combination of the two                                                   

The Bill of Materials for manufacturing an enteral nutritional productin accordance with the present invention is presented in Table 14. It isunderstood that various changes in ingredients and quantities may bemade without departing from the scope of the invention.

                                      TABLE 14                                    __________________________________________________________________________    BILL OF MATERIALS                                                             BATCH SIZE = 45,360 Kgs (100,000 LBS)                                         INGREDIENT                   AMOUNT                                           __________________________________________________________________________    WATER                        31,605.21                                                                          Kgs                                         GUM ARABIC                   437.84                                                                             Kgs                                         ULTRATRACE/TRACE MINERAL PREMIX                                                                            14.50                                                                              Kgs                                         ZINC SULFATE                 2969.89                                                                            gms                                         FERROUS SULFATE              2856.50                                                                            gms                                         MANGANESE SULFATE            784.60                                                                             gms                                         CUPRIC SULFATE               423.11                                                                             gms                                         SODIUM MOLYBDATE             21.39                                                                              gms                                         CHROMIUM CHLORIDE            20.80                                                                              gms                                         SODIUM SELENITE              8.11 gms                                         CITRIC ACID                  894.94                                                                             gms                                         SUCROSE (Carrier)            6520.67                                                                            gms                                         CITRATE                      50.00                                                                              Kgs                                         SODIUM CITRATE               95.00                                                                              Kgs                                         POTASSIUM IODIDE             9.00 gms                                         POTASSIUM CHLORIDE           91.00                                                                              Kgs                                         CORN SYRUP SOLIDS            5630.96                                                                            Kgs                                         MALTODEXTRIN                 1407.52                                                                            Kgs                                         MAGNESIUM PHOSPHATE DIBASIC  131.00                                                                             Kgs                                         CALCIUM PHOSPHATE TRIBASIC   47.50                                                                              Kgs                                         (PREFERABLY MICRONIZED)                                                       CALCIUM CARBONATE            122.50                                                                             Kgs                                         SUGAR (SUCROSE)              852.77                                                                             Kgs                                         FRUCTOOLIGOSACCHARIDE        509.96                                                                             Kgs                                         MEDIUM CHAIN TRIGLYCERIDES   172.69                                                                             Kgs                                         (FRACTIONATED COCONUT OIL)                                                    CANOLA OIL                   99.13                                                                              Kgs                                         SOY OIL                      58.63                                                                              Kgs                                         57% VITAMIN A PALMITATE      250.00                                                                             gms                                         2.5% VITAMIN D               35.00                                                                              gms                                         D-ALPHA-TOCOPHERYL ACETATE (R,R,R)                                                                         10.65                                                                              Kgs                                         PHYLLOQUINONE                6.50 gms                                         30% BETA-CAROTENE            824.00                                                                             gms                                         SOY LECITHIN                 42.64                                                                              Kgs                                         SODIUM CASEINATE             1427.04                                                                            Kgs                                         PARTIALLY HYDROLYZED SODIUM CASEINATE                                                                      1427.04                                                                            Kgs                                         SOY POLYSACCHARIDE           85.28                                                                              Kgs                                         75% WHEY PROTEIN CONCENTRATE 184.46                                                                             Kgs                                         REFINED DEODORIZED SARDINE OIL                                                                             692.87                                                                             Kgs                                         ASCORBIC ACID                37.08                                                                              Kgs                                         45% POTASSIUM HYDROXIDE      25.96                                                                              Kgs                                         TAURINE                      12.00                                                                              Kgs                                         WATER SOLUBLE VITAMIN PREMIX 4.50 Kgs                                         NIACINAMIDE                  1688.60                                                                            gms                                         CALCIUM PANTOTHENATE         1092.24                                                                            gms                                         THIAMINE CHLORIDE HYDROCHLORIDE                                                                            278.78                                                                             gms                                         PYRIDOXINE HYDROCHLORIDE     268.34                                                                             gms                                         RIBOFLAVIN                   217.87                                                                             gms                                         FOLIC ACID                   37.82                                                                              gms                                         BIOTIN                       32.87                                                                              gms                                         CYANOCOBALAMIN               0.75 gms                                         DEXTROSE (Carrier)           882.74                                                                             gms                                         FOLIC ACID                   43.50                                                                              gms                                         CHOLINE CHLORIDE             25.00                                                                              Kgs                                         L-CARNITINE                  7.00 Kgs                                         ARTIFICIAL STRAWBERRY FLAVOR 31.75                                                                              Kgs                                         ARTIFICIAL CREAM FLAVOR      18.14                                                                              Kgs                                         FD & C Red Dye No. 3         1,220.16                                                                           gms                                         __________________________________________________________________________

The liquid nutritional product of the present invention has beenmanufactured by preparing three slurries which are blended together,combined with refined deodorized sardine oil, heat treated,standardized, packaged and sterilized. The process for manufacturing45,360 Kgs (100,000 pounds) of the liquid nutritional product, using theBill of Materials from Table 11, is described in detail below.

A carbohydrate/mineral slurry is prepared by first heating about 6,260Kgs of water to a temperature in the range of about 71° to 77° C. withagitation. The gum arabic is then added to the water using a mixingapparatus. Next the ultratrace/trace mineral premix is added to thewater and dissolved by agitating the resultant solution for at least oneminute. The following minerals are then added, in the order listed, withhigh agitation: Potassium Citrate, Sodium Citrate, Potassium Iodide andPotassium Chloride. The corn syrup solids and maltodextrin are thenadded to the slurry and the temperature of the slurry is maintained atabout 71° C. with high agitation for at least about 20 minutes. Theproduct has been manufactured using maltodextrin distributed by GrainProcessing Corporation, Muscatine, Iowa, U.S.A. under the tradedesignation "Maltrin M-100" and corn syrup solids distributed by GrainProcessing Corporation under the trade designation "Maltrin M-200". Addthe Magnesium Phosphate Dibasic, Calcium Phosphate Tribasic, and CalciumCarbonate to the slurry. The sugar (sucrose), and Fructooligosaccharideare added to the slurry. The product has been manufactured usingfructooligosaccharide powder distributed by Golden Technologies Company,Golden, Colo., U.S.A. under the trade designation "Nutriflora-PFructo-oligosaccharide Powder (96%)". The completed carbohydrate/mineralslurry is held with high agitation at a temperature in the range ofabout 60° to 66° C. for not longer than 12 hours until it is blendedwith the other slurries.

An oil slurry is prepared by combining and heating the medium chaintriglycerides (fractionated coconut oil), canola oil and soy oil to atemperature in the range of about 32° to 43° C. with agitation. The 57%Vitamin A Palmitate, 2.5% Vitamin D₃, D-alpha-tocopheryl acetate (R,R,Rform), phylloquinone and 30% beta-carotene are added to the slurry withagitation. The product has been manufactured using D-alpha tocopherylAcetate distributed by Distillation Products Industries, a division ofEastman Kodak Chemical Company, Rochester, N.Y. U.S.A. under the tradedesignation "Eastman Vitamin E 6-81 D-Alpha Tocopheryl AcetateConcentrate". The soy lecithin is then added to the slurry withagitation. The completed oil slurry is held under moderate agitation ata temperature in the range of about 32° to 43° C. for not longer than 12hours until it is blended with the other slurries.

A protein-and-fiber-in-water is prepared by first heating about 19,678Kgs of water to a temperature in the range of about 60° to 63° C. withagitation. The sodium caseinate, partially hydrolyzed sodium caseinateand soy polysaccharide are blended into the slurry using a mixingapparatus. The product has been manufactured using a partiallyhydrolyzed sodium caseinate distributed by New Zealand Milk Products,Santa Rosa, Calif., U.S.A. under the trade name Alanate 167. Thetemperature of the slurry is lowered to about 57° to 60° C. and then the75% whey protein concentrate is added to the slurry using a mixingapparatus. The completed protein-and-fiber-in-water is held underagitation at a temperature in the range of about 54° to 60° C. for notlonger than 2 hours before being blended with the other slurries.

The oil slurry and the protein-and-fiber-in-water are blended togetherwith agitation and the resultant blended slurry is maintained at atemperature in the range of about 54° to 66° C. After waiting for atleast one minute the carbohydrate/mineral slurry is added to the blendedslurry from the preceding step with agitation and the resultant blendedslurry is maintained at a temperature in the range of about 54° to 66°C. The vessel which contained the carbohydrate/mineral slurry should berinsed with about 220 Kgs of water and the rinse water should be addedto the blended slurry. The refined deodorized sardine oil is then addedto the slurry with agitation. (It is believed that in a most preferredmethod of manufacture the sardine oil would be slowly metered into theproduct as the blend passes through a conduit at a constant rate.) Theproduct has been manufactured using deodorized sardine oil distributedby Mochida International Company, Limited, Shinjuku-ku, Tokyo, Japanunder the trade designation "50% Omega-3 marine oil EPA:DHA 28:12 with0.8% mixed tocopherol as antioxidant". Preferably after at least 5minutes the pH of the blended slurry is determined. If the pH of theblended slurry is below 6.55, it is adjusted with dilute potassiumhydroxide to a pH of 6.55 to 6.8.

After waiting a period of not less than one minute nor greater than twohours the blended slurry is subjected to deaeration,Ultra-High-Temperature (UHT) treatment, and homogenization, as describedbelow:

A. Use a positive pump for supplying the blended slurry for thisprocedure.

B. Heat the blended slurry to a temperature in the range of about66°-71° C.

C. Deaerate the blended slurry to 25.4-38.1 cm of Hg.

D. Emulsify the blended slurry at 61-75 Atmospheres.

E. Heat the blended slurry to a temperature in the range of about 120°to 122° C. by passing it through a plate/coil heat exchanger with a holdtime of approximately 10 seconds.

F. UHT heat the blended slurry to a temperature in the range of about144° to 147° C. with a hold time of approximately 5 seconds.

G. Reduce the temperature of the blended slurry to be in the range ofabout 120-122° C. by passing it through a flash cooler.

H. Reduce the temperature of the blended slurry to be in the range ofabout 71° to 82° C. by passing it through a plate/coil heat exchanger.

I. Homogenize the blended slurry at about 265 to 266 Atmospheres.

J. Pass the blended slurry through a hold tube for at least 16 secondsat a temperature in the range of about 74° to 85° C.

K. Cool the blended slurry to a temperature in the range of about 1° to7° C. by passing it through a large heat exchanger.

Store the blended slurry at a temperature in the range of about 1° to 7°C., preferably with agitation.

Preferably at this time appropriate analytical testing for qualitycontrol is conducted. Based on the test results an appropriate amount ofdilution water (10°-38° C.) is added to the blended slurry withagitation.

A vitamin solution, a flavor solution and a color solution are preparedseparately and then added to the blended slurry.

The vitamin solution is prepared by heating about 394 Kgs of water to atemperature in the range of about 43° to 66° C. with agitation, andthereafter adding the following ingredients, in the order listed:Ascorbic Acid, 45% Potassium Hydroxide, Taurine, Water Soluble VitaminPremix, Folic Acid, Choline Chloride, and L-Carnitine. The vitaminsolution is then added to the blended slurry with agitation.

The flavor solution is prepared by adding the artificial strawberryflavor and artificial cream flavor to about 794 Kgs of water withagitation. A nutritional product according to the present invention hasbeen manufactured using an artificial strawberry flavor distributed byFirmenich Inc., Princeton, N.J., U.S.A. under the trade designation"Art. strawberry 57.883/A" and an artificial cream flavor distributed byFirmenich Inc. under the trade designation "Art Cream 59.200/A". Theflavor solution is then added to the blended slurry with agitation.

A color solution is prepared by adding the FD&C Red Dye No. 3 to about121 Kg of water with agitation. The color solution is then added to theblended slurry with agitation.

If necessary, diluted potassium hydroxide is added to the blended slurrysuch that the product will have a pH in the range of 6.4 to 7.0 aftersterilization. The completed product is then placed in suitablecontainers and subjected to sterilization. Of course, if desired asepticprocessing could be employed.

A method of improving the nutritional status and reversing thecharacteristic diarrhea and inflammatory condition in a mammaliancreature, such as a human, having ulcerative colitis or inflammation ofthe colon comprises enterally feeding to such a mammalian creature, orhuman, a therapeutically effective amount of the nutritional productdisclosed herein.

Clinical trials evaluating the enteral nutritional product disclosedherein in humans will begin in the near future and data supporting thebeneficial properties of the instant invention will be provided. It isexpected that this data will confirm the positive effect of the productdisclosed herein upon ulcerative colitis.

While certain representative embodiments have been described herein forthe purpose of illustrating the invention, it is understood that personsof skill in the art can make various modifications to these illustrativeembodiments without deviating from the scope of the invention.

We claim:
 1. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon by enterally feeding to said mammalian creature a therapeutically effective amount of a liquid nutritional product which comprises:(a) a source of indigestible carbohydrate which is metabolized to short chain fatty acids by microorganisms present in the human colon and which comprises at least one material selected from the group consisting of dietary fibers and indigestible oligosaccharides; and (b) an oil blend containing certain fatty acids, expressed as percentages by weight of total fatty acids in the oil blend, as follows:

    ______________________________________                                         FATTY ACID       % OF TOTAL FATTY ACIDS                                        ______________________________________                                         Oleic acid (18:1n9)                                                                             11.5-15.7                                                     Linoleic acid (18:2n6)                                                                          6.6-9.0                                                       Alpha-Linolenic acid (18:3n3)                                                                   1.5-2.1                                                       Eicosapentaenoic acid (20:5n3)                                                                  15.1-20.5                                                     Docosahexaenoic acid (22:6n3)                                                                   6.3-8.6                                                       ______________________________________                                    


2. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 1 wherein said nutritional product comprises an oil blend containing certain fatty acids, expressed as percentages by weight of total fatty acids in the oil blend, as follows:

    ______________________________________                                         FATTY ACID       % OF TOTAL FATTY ACIDS                                        ______________________________________                                         Oleic acid (18:1n9)                                                                             12.1-15.1                                                     Linoleic acid (18:2n6)                                                                          7.0-8.6                                                       Alpha-Linolenic acid (18:3n3)                                                                   1.6-2.0                                                       Eicosapentaenoic acid (20:5n3)                                                                  16.0-19.6                                                     Docosahexaenoic acid (22:6n3)                                                                   6.7-8.3                                                       ______________________________________                                    


3. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 1 wherein said nutritional product comprises an oil blend containing certain fatty acids, expressed as percentages by weight of total fatty acids in the oil blend, as follows:

    ______________________________________                                         FATTY ACID       % OF TOTAL FATTY ACIDS                                        ______________________________________                                         Oleic acid (18:1n9)                                                                             About 15.2                                                    Linoleic acid (18:2n6)                                                                          About 7.2                                                     Alpha-Linolenic acid (18:3n3)                                                                   About 1.8                                                     Eicosapentaenoic acid (20:5n3)                                                                  About 17.1                                                    Docosahexaenoic acid (22:6n3)                                                                   About 7.7                                                     ______________________________________                                    


4. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to any of claims 1, 2 or 3 wherein, by weight, the ratio of the sum of all of the n-6 fatty acids in the oil blend to the sum of all of the n-3 fatty acids in the oil blend of the nutritional product is in the range of 0.25 to 4.0.
 5. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to any one of claims 1, 2 or 3 wherein, by weight, the ratio of Linoleic acid (18:2n6) in the oil blend to Alpha-Linolenic acid (18:3n-3) in the oil blend of the nutritional product is in the range of 3.0-10.0.
 6. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 4 wherein, by weight, the ratio of Linoleic acid (18:2n6) in the oil blend to Alpha-Linolenic acid (18:3n-3) in the oil blend of the nutritional product i s i n the range of 3.0-10.0.
 7. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 1 wherein the source of indigestible carbohydrates in the nutritional product comprises at least one material selected from the group consisting of gum arabic, soy polysaccharide, fructooligosaccharides, hydrolyzed inulin and xylooligosaccharides.
 8. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 1 wherein the nutritional product further comprises at least one nutrient selected from the group consisting of beta-carotene, vitamin E, vitamin C, taurine and selenium.
 9. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 7 wherein the nutritional product further comprises at least one nutrient selected from the group consisting of beta-carotene, vitamin E, vitamin C, taurine and selenium.
 10. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon by enterally feeding to said mammalian creature a therapeutically effective amount of a liquid nutritional product which comprises:(a) an oil blend which comprises, by weight, a total of at least 25% of one or more oils selected from the group of oils which contain: Eicosapentaenoic acid (20:5n3) and Docosahexaenoic acid (22:6n3); (b) a source of indigestible carbohydrate which is metabolized to short chain fatty acids by microorganisms present in the human colon and which comprises at least one material selected from the group consisting of dietary fibers and indigestible oligosaccharides; and (c) at least one nutrient selected from the group consisting of beta-carotene, vitamin E, vitamin C, taurine and selenium.
 11. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 10 wherein the group of oils in the nutritional product which contain Eicosapentoenoic acid (20:5n3) and Docosahexaenoic acid (22:6n3) consists of fish oils, marine oils, algae oils, fungal oils and vegetable oil.
 12. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 10 wherein the source of indigestible carbohydrates in the nutritional product is a combination of at least one dietary fiber and at least one indigestible oligosaccharide.
 13. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 10 wherein the source of indigestible carbohydrate in the nutritional product contains at least one material selected from the group consisting of gum arabic, soy polysaccharide, fructooligosaccharides, hydrolyzed inulin and xylooligosaccharides.
 14. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon by enterally feeding to said mammalian creature a therapeutically effective amount of a liquid nutritional product which comprises:(a) an oil blend comprising by weight about 5-40% canola oil, about 10-50% medium chain triglycerides, about 25-80% fish oil, about 3-30% soybean oil, and about 2-6% soy lecithin; (b) a source of indigestible carbohydrate which is metabolized to short chain fatty acids by microorganisms present in the human colon and which comprises at least one material selected from the group consisting of gum arabic, soy polysaccharide, fructooligosaccharides and xylooligosaccharides; (c) at least one nutrient selected from the group consisting of beta-carotene, vitamin E, vitamin C, taurine and selenium; and (d) a source of protein.
 15. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 14 wherein the oil blend in the nutritional product further comprises at least one oil selected from the group consisting of corn oil, safflower oil, high-oleic safflower oil, high-oleic sunflower oil, olive oil, borage oil, black currant seed oil and evening primrose oil.
 16. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mammalian creature having ulcerative colitis or inflammation of the colon according to claim 14 wherein, by weight, the ratio of the sum of all of the n-6 fatty acids in the oil blend to the sum of all of the n-3 fatty acids in the oil blend in the nutritional product is in the range of 0.25 to 4.0.
 17. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mamammalian creature having ulcerative colitis or inflammation of the colon according to any one of claims 14, 15 or 16 wherein, by weight, the ratio of Linoleic acid (18:2n6) in the oil blend to Alpha-Linolenic acid (18:3n-3) in the oil blend of the nutritional product is in the range of 3.0 to 10.0.
 18. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mamammalian creature having ulcerative colitis or inflammation of the colon according to claim 14 wherein the nutritional product has about 18.5%-23.5% of total calories provided by protein, about 59.0%-63.0% of total calories provided by carbohydrate, and about 16.0%-20.0% of total calories provided by fat.
 19. A method of improving the nutritional status and reversing the characteristic diarrhea and inflammatory condition in a mamammalian creature having ulcerative colitis or inflammation of the colon according to claim 14 wherein the source of protein in the nutritional product comprises at least one material selected from the group consisting of intact and hydrolyzed proteins of high biological value. 